Abstract: The long term objectives of this proposal are to understand the basis for the high fidelity exhibited by replicative DNA polymerases (DNA pols). Specifically we want to: (i) understand how the dynamics of conformational changes exhibited by DNA pols affect base discrimination; (ii) follow structural changes in DNA pols and their fidelity mutants during DNA synthesis with correct (R) and incorrect (W) incoming dNTPs; (iii) identify transient intermediates in the reaction pathway for primer extension with R and W dNTPs using wild type (wt) and fidelity mutants of DNA pols; (iv) identify structural features of the RB69 pol fidelity mutants that are responsible for the high level of misincorporation exhibited by these mutants; (v) identify the chemical features of a nascent base-pair that allow the incoming dNTP to be inserted with high efficiency. To reach these goals we will determine rates of conformational changes when the wt and mutant pols encounter dNTPs that harbor either non-complementary bases or nucleobase analogs. For this purpose we will employ; (i) stopped-flow fluorescence with dye labeled polymerases and with fluorescent base analogs in the template strand of primer-templates (P/Ts) in ensemble-averaged and in single-molecule experiments; (ii) Single-Molecule Fluorescence Energy Transfer (smFRET) to investigate the dynamics of the nucleotide addition cycle with the aim of capturing transients in the reaction pathway; (iii) rapid chemical quench experiments to obtain rates of product formation; (iv) X-ray crystallography to determine structural changes that occur when wt dNTPs or dNTPs containing base analogs are bound in ternary complexes (v) polychromatic, time-resolved X-ray crystallography using Laue diffraction to visualize changes in the structure of intermediates during the nucleotidyl transfer reaction. This can be accomplished by using caged dNTPs that can be converted to dNTP substrates by photolysis. We are using RB69 pol, a member of the B family (which includes two human DNA pols, pol alpha and pol delta) so that information acquired from RB69 pol and its mutants should be relevant to human DNA pols as well. Taken together, the results of these investigations will contribute to a better understanding of diseases that involve malfunctioning of DNA pols required for replication and repair. They will also provide the basis for devising therapeutic strategies to curtail or halt DNA replication and/or repair which could cripple malignant cells. This information could also be helpful in combating infections caused by viruses, bacteria or parasites.